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Lipkin finds biomarkers not bugs

The CDC PCOCA telephone broadcast on 10 September 2013, featured a lengthy presentation from Dr Ian Lipkin who revealed some stunning initial results from the study that is primarily hunting for pathogens in ME/CFS. Simon McGrath and Russell Fleming (Firestormm) review this exciting and possibly game-changing news...

He started off by lowering expectations: their results thus far do not implicate a single infectious agent and they have not discovered the cause of CFS. But he then wowed the hundreds listening to the conference call by revealing they had found strong evidence for immune overstimulation, both in blood plasma and in cerebro spinal fluid; and that they were now hard at work trying to identify what could be causing these abnormalities.

The big pathogen hunt draws a blank thus far

This study is looking exhaustively for any pathogen: viral, bacterial, fungal or parasitic, to see if a chronic infection could explain ME/CFS. Many of the results are in, but so far there is no clear sign of viruses at least. It is every bit a collaborative effort, with patients provided by long-established physicians including Dr Dan Peterson and Professor Jose Montoya.

The Key Players

Lipkin began by thanking the clinicians who had provided the all-important samples. Effectively it was a roll call of America's top ME/CFS physicians:

He went on to acknowledged the very necessary support of the Chronic Fatigue Initiative, the Evans Foundation and an anonymous donor supporting Montoya's work.​

First the researchers looked at blood plasma for 285 CFS patients and 201 controls from Jose Montoya at Stanford (these are serious numbers). They searched for genetic evidence of infection using a panel that was able to detect 20 specific bacteria, viruses or parasites; including Epstein-Barr virus, Human Herpes Virus 6, enteroviruses, Influenza A and Borrelia bacteria.

But they effectively drew a blank using both methods - only 1.4% of CFS patients were found to have HHV6 infection, but so did 1% of controls.

Other findings...

Lipkin said they also found Anelloviruses in 75% of those studied in plasma samples, but it was not specific for Chronic Fatigue Syndrome (we don't currently have separate figures for patients and controls). Anellovirus is believed to cause widespread chronic infection in the human population, but has not yet been associated to any specific disease. "I really don’t know at this point what this finding means" said Lipkin.

They also found retroviruses in 85% of the sample pools. However, Lipkin expressed caution:

"It is very difficult at this point to know whether or not this is clinically significant. And given the previous experience with retroviruses in Chronic Fatigue, I am going to be very clear in telling you – although I am reporting this at present – in Professor Montoya’s samples neither he nor we have concluded that there is a relationship to disease ...if I were to place bets and speculate, I would say that this is not going to pan out."​

Window on the brain: Cerebrospinal fluid samples

Both Lipkin and Hornig were clearly excited to have had access to the 60 cerebrospinal fluid samples from Peterson, and kindly donated by his ME/CFS patients. Cerebrospinal fluid bathes our brain and spinal cord so it gives access to what goes on in the brain, a prime site of interest to researchers, but hard to access; so these spinal tap samples are precious.

However, using the same molecular techniques to track down pathogens as they had applied to Jose Montoya's samples, Lipkin's team again drew a blank.

At the Invest in ME conference in May, Hornig said they had tentative findings that there might just be a novel pathogen or pathogen candidate in these samples - but it was too early to say for sure. In reply to a question yesterday, Lipkin said there is no more to report:

"we do not yet have a novel pathogen nor do we have a pathogen candidate in which I would have any confidence. So at this point we have no plans to publish anything."
​

Nonetheless, he also said they hadn't finished all the DNA sequencing work on these samples, so this is not yet a dead end.

Immune stimulation and biomarkers

Up to this point we'd heard a lot of very impressive negative findings, based on large, well-defined samples of patients and controls, and using powerful research techniques. But what we most wanted to hear about was positive leads - and Ian Lipkin duly obliged:

Plasma is the clear fluid left when blood is separated​

They took plasma from the CFI cohort of 200 very well-defined ME/CFS cases and 200 controls collected by clinicians (See 'Key Players' above). Then they searched for cytokines and chemokines, which are regulators and controllers of the immune system.

The big finding was a decrease in levels of Interleukin's IL-17, IL-2, IL-8 and in TNF-alpha - all of which have a role in pro-inflammatory responses. Lipkin believes these significant differences are worth follow-up in larger cohorts to better understand what they mean.

Lipkin and colleagues also found upregulation in Leptin, a hormone that plays a key role in regulating energy uptake and use, and Serpin, a protein family with multiple roles. More obscure cytokines, and and their cousins chemokines, were also affected but Lipkin didn't give any further detail.

Two new patient types revealed

Ian Lipkin said they found something very surprising in their data: there appear to be substantial differences in profiles between those people who have had the disease for 3 years or less and those who have had the disease for more than 3 years. And that this is important because, "it could have implications for therapy as well as for diagnosis".

In the ‘early group’, who have been ill for less than 3 years, there seem to be a number of markers suggestive of some sort of allergy aspect. For example there are often increased numbers of eosinophils in blood, with more differences in cytokines. But while this is tentative, he was more confident in the finding the IL-17 was elevated in these 'early' patients, compared with reduced levels in patients ill for over three years.

Lipkin said that he thought this 3-year division could be very important and that it hadn't been something they were looking for; but would be looked at in any future work they did.

Cerebrospinal fluid: different differences

Lipkin's team also found differences in cerebrospinal fluid biomarkers between patients and controls. I'm not sure from what he said if there were significant differences between the 'early' group and patients who had been ill for more than three years. However, they did find that patients had elevated levels of the TH-2 type cytokines IL-10 and IL-13 and elevated levels of four TH-1 cytokine: IL-1 beta, TNF-alpha, IL-5, and IL-17. Lipkin said this is compatible with a profile of some particular types of response that may provide insights into immunological dysregulation in Chronic Fatigue Syndrome.

So, overall they found clear differences between patients and controls in both plasma and spinal fluid. Many immune differences have been detected before, but findings are not very consistent and none had used such a large and robustly-defined patient cohort (meeting Fukuda and Canadian criteria), or with such carefully matched controls.

HEALTH WARNING​

Dr Ian Lipkin again was very clear that he did NOT recommend anyone act on these provisional findings: he doesn't want anyone to get a spinal tap, or measure the cytokine levels and hope to modulate them with drugs based on this research: it's too early for that.​

​

The lost years...

​

Back in 1999 Ian Lipkin published his first paper on CFS, which ruled out a connection between the illness and Borna Disease Virus (one of the many viruses he's discovered). But crucially they found evidence of polyclonal B-cell reactivity. He commented that back then:

"there was a very strong sentiment in some portions of the scientific and clinical communities – not always and not everywhere – but in some portions of the community, that this was a psychological illness. What I said was that based on our findings we had very strong evidence that people with Chronic Fatigue Syndrome were truly ill with a physical illness and they deserved a "Deep Dive" to find out why they were ill." ​

​

He noted that was a long time ago and dryly added "I am pleased to see that people are now paying much more attention to this disorder and what we can do about it".

​

What next?

"I still believe the primary cause is likely to be an infectious agent."
​

Having found clear signs of cytokine abnormalities, Lipkin wants to find out what is causing these changes; he currently thinks that an ongoing stimulus is causing immune system activation which might be resulting in the symptoms we associate with the disease. And interestingly, he still believes that infection lies at the root of it all - despite not yet finding any infections.

The team - working closely with the clinicians he's already mentioned - plan even more extensive work on a cohort of 200 CFI patients and controls. They will further test for viruses and crucially they will also look for bacteria and fungi by sequencing ribosomal RNA (which should detect unknown bacteria and fungi, as well as known ones).

Previous infection detection

The group has looked for current pathogens in the blood and spinal fluid samples they have - and has so far drawn a blank, though there is more work underway. However, what if the trigger for ME/CFS is an earlier infection that has since been cleared: the 'hit and run' scenario Hornig has described? Lipkin says they are looking for "shadows" of previous infections by searching for antibodies against specific infections, rather than the infections themselves (the body will continue to produce low levels of specific antibodies years after an infection has been cleared (the basis of much immunity)).

They will use a technique called LIPS to look for antibodies against particular known infection. In addition, as in their viral sequencing work, they will use an 'unbiased' system (using peptide arrays) that should detect all potential viruses - or rather antibodies against them. They don't currently have an 'unbiased' system that works on bacteria or fungi.

Fecal future?

What excites Ian Lipkin most is the gut microbiome, the community of micro-organisms that live in our gut - which might seem odd given his focus so far on pathogens and the immune system. However, it turns out that the microbiome can have a profound influence on the immune system and may be important in many diseases. Hornig and Lipkin have already developed techniques to study the microbiome, and have shown it could be a factor in autism and even in the severity of colon cancer.

It turns out the best way to find out what lives in the microbiome is to measure what comes out of the gut: fecal matter, or 'poop' to you and me. Their ME/CFS microbiome project has started, but Lipkin felt there wasn't enough material in the first attempt at gathering samples, so things have been delayed as they switch to a new technique (the 'special' collection cups).

The Big Ask: "we can't do this without you"

Microbiome research is very expensive (analysis, not sample collection), and they currently only have the funds to do 10% of what's needed. In fact, throughout the call, Lipkin was stressing the need for more funds, because good research costs and ME/CFS is woefully underfunded: there just isn't enough money to do the work needed. The current science budget cuts (Sequestration) in the US makes the climate even harder.

"It is probably inappropriate for me to be passing the hat, but that’s precisely what I am doing."​

He urged patients to contact their representatives in Congress, demanding more funding for ME/CFS research - pointing out that in the early days of HIV, there was little funding until patients demanded it. He also said that if patients were able to afford it, they should invest in research themselves.

Conclusion

ME/CFS research is coming of age. Ian Lipkin has reported these early - and soon-to-be published results - from a huge study, using 285 of Jose Montoya's patients, the 200 patients from the Chronic Fatigue Initiative, plus the 60 'gold-dust' spinal fluid samples from Dan Peterson's patients. Using state-of-the-art techniques, they have largely ruled out a role for a specific pathogen - though that work is incomplete. And they have strong evidence of ongoing immune stimulation in ME/CFS patients, with abnormalities found in both the blood and spinal fluid. The next stage involves deeper searching for bacteria and fungi, looking for 'shadows' of previous infections and investigating the gut microbiome. These are exciting times!

Thanks to the CDC for organizing the presentation and to Firestormm for providing a transcript of this talk, and above all to Dr Ian Lipkin for taking time out to talk to patients.

If you would like to donate to Ian Lipkin's research, click here. In the comments box, put "for M.E/C.F.S Study" to make sure it goes to the right place.

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Great article guys! Exciting to hear about the signs of immune activation and also interesting to me to hear about the increase in Leptin, i have to admit I overlooked it intially but reading the article it stand out to me as a very interesting upregulation to see. It makes a lot of sense to have upregulation of a molecule such as this given the evidence of mitochondrial and ATP production problems!

I think it's worth mentioning too that these findings given important evidence for both the autoimmune theory that Fluge and Mella are working on and indeed the pathogen theory that Lipkin himself believes. Hopefully some of these abnormalities hold up to further scrutiny!

...interesting to me to hear about the increase in Leptin, i have to admit I overlooked it intially but reading the article it stand out to me as a very interesting upregulation to see. It makes a lot of sense to have upregulation of a molecule such as this given the evidence of mitochondrial and ATP production problems!

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Thanks!

Re Leptin:

the blog said:

Lipkin and colleagues also found upregulation in Leptin, a hormone that plays a key role in regulating energy uptake and use

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Yes, I was intrigued by that too, though I that line is pretty-much the extent of my knowledge of Leptin.

I think it's worth mentioning too that these findings given important evidence for both the autoimmune theory that Fluge and Mella are working on and indeed the pathogen theory that Lipkin himself believes. Hopefully some of these abnormalities hold up to further scrutiny!

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Yes, and I think they key thing is replication, which is very much what Ian Lipkin plans to do. I have to say, these broad findings are probably consistent with many different theories so I will be very interested in what the future work turns out: certainly Ian Lipkin's main focus now seems to be to find what is responsible for these changes.

This is remarkable. I think it's interesting that this study makes ME/CFS somewhat similar to MS, particularly in the "hit and run scenario". A virus-like infection comes in shortly, disrupts the immune system, and it's all downhill from there.

Also, how do we pressure for more research funds? How do we organize and lobby to get these researchers what they need?

Indeed it has but we now seem to be getting to the bare bones of the issue with what exactly is wrong with strong evidence to support these initial claims - if nothing else these findings are good clues as of where to look next!

Sometimes finding nothing at all is the best outcome. In terms of viruses as a central causation, this somewhat conclusively shows little significance in the commonly held perpetrators which means we can move on to newer ideas without the baggage that these old ideas bring with them.

An analogy I made in a previous thread earlier today seems to also be relevant here I feel with regards to the 'hit and run' idea many people discuss. The reference to autoimmunity is however just a personal opinion I hold and for the purpose of this topic should be interchangeable with immune activation.

As an analogy I believe the genetic predisposition is like having petrol liberally doused over an area - it's invisible (but likely a few small hints that go unnoticed) - it then only takes a spark in the form of a viral or bacterial trigger to start the immune activation and through exploitation of the genetic predisposition and suddenly a whole fire breaks out - this is when the symptoms first appear and suddenly it becomes much more difficult to manage. There is little point in identifying the initial virus or bacterial 'spark' as this does nothing to ease the fire. The only option then comes in calming the aberrant immune response and trying to break the autoimmune cycle.

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The interesting part here is the specifics of the immune activation - certainly not all of these will prove to be repeatable but if just a handful do we could be looking at a good trace of why the immune response has turned so aberrant.

Thank you Simon and Firestormm for the hard work putting this clear review together and supplying a transcript for us. I had listened in to the meeting but, apparently missed a lot of the details.

It is clear to me that Lipkin found enough abnormalities/biomarkers for him to be excited to plunge in deeper for further studies. Any study showing differences between patients and controls is promising. It remains to be shown/proven what this will lead to.

Their finding of the three year marker showing differences in the results is intriguing. I wonder why three? As he stated though, they were not looking for that, it just has seemed to appear when comparing results.

I wish I could understand in layman's terms what these abnormalities in cytokines mean or what this Leptin issue means.
I almost wish I had gone through medical school in order to understand and follow these developments.

Great news! Thanks for the article. I truly hope that they advance the understanding of the disease mechanisms. It's fascinating that some inflammatory markers were even low. I also hope that microbiome analysis gets a lot cheaper through better implementation of computers and resulting automation.

In addition to regulating the hunger-response, Leptin controls the release of toxins from the fatty tissues.

Lipkins findings of Leptin is consistent with "the elusive biomarker for CFS" that we have known about for about fifteen years now.

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With regard to Leptin, I think it is a very interesting find and it's more interesting that it has been found in the past. I'm not sure it will prove to be a causative agent in the disease pathology of ME but certainly it would be interesting to know why it appears to be getting up-regulated - I suspect it could be due to its role in metabolism considering the findings of dysfunctional mitochondria and Lactate build-up in the muscles - presumably through increased anaerobic respiration during trivial exertion.

As you might expect....It's complicated.
Leptin stimulates MSH "Melanocyte Stimulating Hormone"
Which in turn regulates the innate immune system.
High Leptin and low MSH is seen in illnesses where toxins are stored in the fatty tissues.
The high Leptin makes weight loss extremely difficult.

Since CFS researchers have yet to make the connection between this phenomenon and CFS, the connection remains to be explored. But for Lipkin to see it makes it more likely that CFS researchers will take an interest.

I wonder if the genes associated with Leptin will be determined to play any role. For now I checked three of the SNPs associated with Leptin and I have the common variety so they dont explain my expereince. I am one of the 'fighting this battle longer than three years' group. I still suspect when I had mono in 1995 that was the start of it all even though I did not recognize the long periods between crash and recovery as being related. The last 6-7 years I know realize the unmistakable relation. I am just coming out of another crash (6-7 weeks) and there is a difference, more function (although not sure I'll ever get back to 100%), not as sick all the time, symps not as severe. I wish I understood this crash and recovery element to whatever is causing this . Do others also experience this crash and recovery I am describing? There are some symps I have all the time but when I crash I am more disabled, other symps added and the ones I usually have seem worse then like a cloud lifting I enter a day or week that I realize I am much better. It is so friggen madding the inconsistency. I blame that most for the reason I lost my career, how can I or my employer count on me when I dont know one day to the next if I can function. Sorry for the derail I just want answers, to heal and get my life back!!!!

It seems that he did find bugs. He reported that he found both anellovirus sequences and also retroviral sequences. He said he didn't know what it meant of if it would lead anywhere. Given the craziness that happened last time a researcher tried to associate a viral cause with CFS I don't blame him for being cautious and wanting more evidence before making any claims. But he did state he felt the cause still seemed like an infection and that all of the findings (bugs or not) should be followed up.

Also, I have a hard time reconciling the claims about IL-8. Several other studies have shown IL-8 to be significantly elevated in patients with CFS. My own IL-8 is one of the most consistently elevated markers, and I have participated in several of these studies through my CFS doctor (including this one). I'll admit though, I don't know the intricacies of testing for cytokines. Perhaps they vary based on time of day, or processing method or something.

It seems that he did find bugs. He reported that he found both anellovirus sequences and also retroviral sequences. He said he didn't know what it meant of if it would lead anywhere. Given the craziness that happened last time a researcher tried to associate a viral cause with CFS I don't blame him for being cautious and wanting more evidence before making any claims. But he did state he felt the cause still seemed like an infection and that all of the findings (bugs or not) should be followed up.

Also, I have a hard time reconciling the claims about IL-8. Several other studies have shown IL-8 to be significantly elevated in patients with CFS. My own IL-8 is one of the most consistently elevated markers, and I have participated in several of these studies through my CFS doctor (including this one). I'll admit though, I don't know the intricacies of testing for cytokines. Perhaps they vary based on time of day, or processing method or something.

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Acer2000:

You bring a very good point. I noticed he mentioned the anellovirus and traces of some retrovirus, but qualified it quickly as needeing more research. After the xmrv debacle, you can't blame any researcher for being utterly cautious.

I think the title is not necessarily misleading, but leaning heavily on Dr Lipkin's cautious approach to the findings.

Back in 1999 Ian Lipkin published his first paper on CFS, which ruled out a connection between the illness and Borna Disease Virus (one of the many viruses he’s discovered). But crucially they found evidence of polyclonal B-cell reactivity. He commented that back then:

“there was a very strong sentiment in some portions of the scientific and clinical communities – not always and not everywhere – but in some portions of the community, that this was a psychological illness. What I said was that based on our findings we had very strong evidence that people with Chronic Fatigue Syndrome were truly ill with a physical illness and they deserved a “Deep Dive” to find out why they were ill.”​

​

He noted that was a long time ago and dryly added “I am pleased to see that people are now paying much more attention to this disorder and what we can do about it”.

Click to expand...

Dr. Lipkin is even more emphatic on this earlier in the call...

"My own interest in Chronic Fatigue Syndrome dates back to the mid-to-late 1990’s when I was working with Brigette Evangard, and one of the questions that came in earlier today had to do with polyclonal B cell activation. Those of you who may have read that paper that I wrote with Brigettea, which was ultimately published in 1999, was one of the first to really draw attention to the fact that people with Chronic Fatigue Syndrome who were diagnosed using the criteria at the time did in fact have a physical condition.

"Although we were unable to find evidence of infection with borna disease virus, which was the primary question at that point, we did demonstrate unequivocally that the vast majority of people who met these criteria had polyclonal B cell activation, so they clearly had some sort of immune activation. But the question was, why?"

Montoya's recently released results from a small (10 participants, I believe) study: They obtained blood draws for 25 consecutive days from these patients, who also, I believe, kept journals indicated their fatigue levels. The only finding that correlated directly with fatigue levels in most (not all) of these patients was the hormone leptin. I think that the overall levels were not abnormal, but the changes in leptin tracked the fatigue level changes.
I imagine the details of this are discussed somewhere in the forum, but I haven't had a chance to track it down yet.